Transcription and DNA methylation dynamics during bovine oocyte growth

During oogenesis, tightly orchestrated molecular and structural modifications occur in the nucleus and cytoplasm of oocytes which are fundamental to successful gametogenesis and development, but susceptible to modulation by environmental cues. Within the nucleus, several events are included in the large-scale chromatin configuration and remodeling, such as DNA methylation, nucleolus reorganization, and the establishment of transcription. Several studies demonstrated epigenetic changes in mice and human oocytes. However, less information is known about methylome and transcriptome during bovine oocyte growth. Therefore, the present project aims to create a molecular and morphological compendium of bovine oocyte ontogeny, revealing the epigenomic and transcriptomic signature of early antral to antral follicle oocytes. By applying cutting-edge technology, such as single-cell Genome and Transcriptome sequencing in growing bovine oocytes we identified clusters of genes with expression positively correlated to oocyte size, including genes related to cell division, ubiquitin-dependent catabolic process, mRNA processing, and microtube cytoskeleton organization, among others. On the other hand, clusters negatively correlated to oocyte size included genes related to T cell receptor signalling pathway, regulation of transcription from RNA polymerase II promoter, signal transduction, and positive regulation of gene expression, among others. Differential expression analysis was also performed between consecutive oocyte size groups ranging from <70 to >120 μm in diameter. Oocytes from secondary follicles (<70 μm in diameter) express genes associated with ribosome biogenesis, mitochondrial function and ATP production. Whereas expression of maternal-effect genes was upregulated in oocytes from multi-laminar preantral follicles (70-79 μm in diameter). In line with the high cytoplasmic activity of oocytes from early antral follicles (80-99 μm in diameter), the expression of genes related to the regulation of transcription, intracellular transport, and proteasome assembly was increased. Reflecting oocyte acquisition of meiotic competence towards the end of the growth phase, nuclear pathways including meiotic nuclear division and chromosome segregation were upregulated, whereas those associated with cytoplasmic pathways were downregulated in oocytes >100 μm in diameter. Oocytes from dominant follicles presented a similar gene expression, with only oxidative phosphorylation being upregulated at certain time points, showing the importance of energy production during oocyte maturation. From the single-cell bisulphite conversion, global CpG methylation revealed a similar percentage in oocytes <90 μm in diameter, constantly increasing until >120 μm oocytes, indicating the onset of methylation in oocytes from mid-antral follicles. Ovary tissue slides were used for immunohistochemistry to detect 5mC, indicating partial chromatin staining in most of the oocytes from pre-antral follicles, which differs from the pattern presented in oocytes from antral follicles. Fluorescence measurement was not different in oocytes from primordial until secondary follicles, and it significantly increased in oocytes from antral follicles, confirming our hypothesis. These results are in agreement with previous global CpG methylation from the bisulphite conversion, showing an increase in DNA methylation across oocyte growth. In conclusion, the present study brings novel information on molecular modifications in growing bovine oocytes, contributing to the understanding of this dynamic phase of gamete development.